Biocontrol of Rhizoctonia solani, the causal agent of bean damping-off by fluorescent pseudomonads.

Rhizosphere bacteria belonging to the fluorescent pseudomonads are receiving increasing attention for the protection of plants against soil-borne fungal pathogens. Among these pathogens, Rhizoctonia solani, the causal agent of bean damping- off is very important in bean fields of Iran. In this study, the antagonistic activity of 46 isolates of fluorescent pseudomonads (isolated from different area of Iran) and Pseudomonas fluorescens strain CHA0 investigated against one isolate of R. solani. About 64% of isolates revealed antagonistic activity against R. solani. Production of antifungal metabolites such as HCN, siderophore and protease was evaluated. The results showed that 97.8%, 17% and 78% of isolates produced siderophore, HCN and protease respectively. There was no significant correlation between antagonistic activity and production of these metabolites. Isolates P-5, P-10 and P-32 with strain CHA0 were selected in order to investigate involvement of siderophore, volatile metabolites (HCN), and non-volatile metabolites in reducing mycelial growth of R. olani. Isolate P-5 showed much more inhibitory effect by production of volatile metabolites and siderophore. Non-volatile metabolites in isolates P-32 and P-5 completely inhibited mycelial growth of the fungus. After the primary labrotory tests, isolates P-14, P-35, P-30, P-5 and strain CHA0 were selected for in vivo experiments. These selected isolates with benomyl fungicide were used as seed coating and soil drenching in sterile soil under greenhouse condition. The result indicated that in seed treatment method, isolates P-30 by 66% had the most effect in disease reduction while in soil treatment method, strain CHAO by 60% had the most effect, such that this two isolates showed significant differences in comparison with plants inoculated with R. solani inoculums.

Contribution of phlA and some metabolites of fluorescent pseudomonads to antifungal activity.

Fluorescent Pseudomonas species are an important group of PGPR that suppress fungal root and seedling disease by production of antifungal metabolites such as 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin, pyrolinitrin, siderophores and HCN. The compound 2,4-DAPG is a major determinant in biocontrol of plant pathogens. A 7.2 kbp chromosomal DNA region, carrying DAPG biosynthetic genes (phlA, phlC, phlB, phlD, phIE and phlF). Detecting the ph1 genes make them an ideal marker gene for 2,4-DAPG-producing fluorescent pseudomonad's. In this study we detected ph1A gene (that convert MAPG to 2,4-DAPG) using PCR assay with primers phlA-1r and phlA- f that enabled amplification of phlA sequences from fluorescent pseudomonad's from ARDRA group 1 and 3. We could detect phlA gene in P. fluorescens strains CHAO, Pf-44, Pf-1, Pf-2, Pf-3, Pf-17, Pf-62 and Pf-64, native isolates of Iran. The efficacy of this method for rapid assay characterizing rhizosphere population of 2,4-DAPG producing bacteria from soil of different area of Iran is in progress. We used a collection of 48 fluorescent pseudomonas strains in vitro, with known biological control activity against some soil born phytopathogenic fungi such as, Macrophomina phaseoli, Rhizoctonia solani Vericillium dahlia, Phytophthora nicotiana, Pythium spp. and Fusarium spp. and the potential to produce known secondary metabolites such as protease. Strains Pf-1, Pf-2, Pf-3, Pf-17, Pf-33 and Pf-44 showed the best antifungal activity against all fungi used in this study. Thirty-eight of 48 strains produced protease. The ability to rapidly characterize populations of 2,4-DAPG producers will greatly enhance our understanding of their role in the suppression of root disease.